The p53 gene is the most commonly mutated tumor suppressor gene in human cancers. p53 plays multiple roles in cellular responses to various stresses, including inducing cell cycle arrest or apoptosis. p53 functions as a transcription factor and has a sequence-specific DNA-binding domain in its central region, a transcriptional activation domain at its amino (N) terminus and a tetramerization domain at its carboxyl (C) terminus. In response to DNA damage and other cellular stresses, the protein levels and activity of p53 as a transcription factor are greatly increased. Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxy terminus by CBP/p300 in response to genotoxic and some non-genotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317 to Arg (K317R) missense mutation into the endogenous p53 gene. p53 accumulated to normal levels in p53K317R mouse embryonic fibrblasts (MEF) and thymocytes after DNA damage. While p53-dependent cell cycle arrest was normal in p53K317R MEFs after DNA damage, increased p53-dependent apoptosis was observed in p53K317R thymocytes, small intestinal epithelial cells and retina after ionizing radiation (IR). Consistent with these findings, p53-dependent gene expression was significantly increased in p53K317R thymocytes after IR. The increased p53 apoptotic activities correlate with higher sequence-specific DNA-binding activity to p53-dependent promoters in p53K317R thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities by modifying its sequence-specific DNA binding activity. Phosphorylation of p53 is known to play important roles in the regulation of p53 stability and activity. To understand the mechanism whereby p53 stability and activity are regulated during retinoic acid (RA)-induced embryonic stem (ES) cell differentiation, we profiled the phosphorylation pattern of p53 in ES cells during RA-induced differentiation. For this analysis we utilized ES cells containing a humanized version of mouse p53, denoted humanized p53 knock-in (p53hki). Phosphorylation of p53 at Ser315 appeared to be the major phosphorylation event during RA-induced ES cell differentation. Moreover, p53 bound to the promoter of Nanog, a homeodomain protein exclusively expressed in (ES) cells that is required for ES cell self-renewal, and suppressed Nanog expression after DNA damage. The rapid downregulation of Nanog mRNA during ES cell differentiation correlated with the induction of p53 transcriptional activity and Serine 315 phosphorylation. In addition, the induction of p53 activities was impaired in p53K315A knock-in ES cells during RA-induced differentiation, leading to inefficient suppression of Nanog expression and resistance to RA-induced differentiation. We found that the decreased inhibition of Nanog expression in p53K315A ES cells during differentiation was due to an impaired interaction between p53K315A and the co-repressor mSin3a, leading to reduced recruitment of mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ES cells, by inducing the differentiation of ES cells into other cell types that undergo efficient p53-dependent cell cycle arrest and apoptosis. The wildtype p53-induced phosphatase (Wip1 or PPM1D) is a serine/threonine phosphatase that is transcriptionally upregulated by p53 following UV and ionizing radiation. PPM1D inhibits p53 activity indirectly through dephosphorylation and downregulation of the p53-activating p38 MAP kinase. PPM1D is an oncogene in transformation assays and is amplified or overexpressed in several human tumor types. Last year, we demonstrated that PPM1D interacts with the nuclear isoform of uracil DNA glycosylase, UNG2, and suppresses base excision repair (BER). Point mutations that inactivate PPM1D phosphatase activity abrogate BER suppression, indicating that dephosphorylation by PPM1D is important for suppression of BER. We have identified UNG2 phosphorylation sites at threonines 6 and 126 that exhibit enhanced phosphorylation following UV irradiation. UNG2 phosphothreonine 6 is dephosphorylated in vivo by PPM1D. PPM1D may thus function in BER by binding and dephosphorylating the activated form of UNG2 to return it to a homeostatic state following repair of DNA damage. The effects of PPM1D on DNA repair have implications for its role as an oncogene. Overexpressed PPM1D in cancer cells may suppress BER and perhaps other forms of DNA repair, increasing the likelihood of further oncogenic mutations. In fact, we have found preliminary data in cancer cell lines that support this possibility. MCF-7 breast cancer cells, with very high levels of PPM1D, have much lower BER activity compared to cancer cell lines that have normal levels of PPM1D. Thus, overexpression of PPM1D in tumors could increase genomic instability both through relaxed checkpoint control (via inactive p53) and through decreased DNA repair efficiency.